HGF/SF (hepatocyte growth factor/scatter factor) is a multifunctional heterodimeric polypeptide produced by mesenchymal cells, and it is composed of an alpha-chain containing an N-terminal domain and four kringle domains (NK4) covalently linked to a serine protease-like beta-chain C-terminal domain. Further, human HGF/SF is produced in the form of a biologically inactive single chain precursor consisting of 728 amino acids, and biologically active HGF/SF is generated therefrom through cleavage at the R494 residue by a specific serum serine protease. The active form of HGF/SF is a disulfide-linked heterodimer composed of a 69 kDa alpha-chain and a 34 kDa beta-chain.
The binding of HGF/SF to its receptor, CMET, induces the growth of various cell types, mediates the epithelial mesenchymal transitions and the formation of tubules and lumens, and promotes angiogenesis. A cMET and HGF/SF knockout mouse is not often embryonically viable and shows developmental defects in placenta, fetal liver and limb/muscle formation (Cao et al., PNAS 98 (13): 7443-7448, 2001; Gmyrek et al., American Journal of Pathology 159 (2): 579-590, 2001). Further, cMET was reported to be over-expressed in various human cancers of the liver, prostate, colon, breast, brain and skin (Maulik et al, Cytokine & Growth Factor Reviews, 13(1), 41-59,200). cMET activation markedly enhances the metastastic spread of cancer because of its stimulatory influences on the processes e.g., angiogenesis, cell motility, and cell surface protease regulation, and therefore, cMET has been regarded as an important target factor for the prevention and treatment of cancer (Wielenga et al., American Journal of Pathology 157 (5): 1563-1573, 2000).
Monoclonal antibodies (mAbs) have enormous potential as therapeutic agents. However, non-human antibodies are highly immunogenic in the human body and their short serum half life severely limits their clinical efficacy. To circumvent such a problem, a “humanized antibody” has been constructed by grafting complementarity-determining regions (CDRs) of the parent mAb variable region that directly binds to antigens onto a human antibody framework. (CDR-grafting method). The humanized antibody thus obtained reduces the immunogenicity in human, and prolongs the serum half-life in human while keeping the binding affinity and specificity of the parent antibody (Baselga, J. et al., J Clin Oncol, 14, 737-744, 1996).
However, the simple CDR-grafting method often yields humanized antibodies which bind to their antigens much more weakly than the parent mAb (Carter, P. et al., Proc Natl Acad Sci USA, 89, 4285-4428, 1992; Eigenbrot, C. et al., Proteins, 18, 49-62, 1994; and Kettleborough, C. A. et al., Protein Eng., 4, 773-778, 1991)
To provide a humanized antibody which preserves the original affinity of the parent mAb, the key residues in the framework regions (FRs) of the human antibody essential for forming a CDR loop were replaced with the corresponding amino acid residues from the parent mAb (Chothia, C. et al., Nature, 342, 877-888, 1989). But, the identification of such framework residues that affect antigen binding has proved to be unduly time-consuming.
The present inventors sought to develop an anti-HGF/SF humanized antibody having the equal or greater binding activity than that of the parent antibody with reduced immunogenicity in human.